Primary reactive oxygen species (ROS) such as superoxide radical, hydrogen peroxide, hydroxyl radicals, and ortho-quinone derivatives of catecholamines exert their cellular effects by modifying DNA, lipids, and proteins to form secondary electrophiles. Examples of such latter secondary electrophiles include hydroxyalkenals, nucleotide propenals, and hydroxyperoxy fatty acyl chains. The secondary electrophiles are implicated in cellular dysfunction either because they are no longer able to participate in normal cellular activity or because they serve as electron acceptors in oxidative chain reactions that result in the modification of other essential cellular components. Damage caused by the primary and secondary ROS contributes to the pathogenesis of important human disease caused by neuronal ischemia during stroke, post-cardiopulmonary bypass syndrome, brain trauma, and status epilepticus. ROS likely participate in cardiac damage induced during ischemic heart disease, renal damage induced by ischemia and toxins as well as in more chronic diseases such as the destruction of the islets of Langerhans of the endocrine pancreas in Diabetes Mellitus, the destruction of neurons in Parkinson's disease, and other chronic neurodegenerative disorders.
One way that cells handle the deleterious effects of ROS is through a preconditioning response. The preconditioning response is an adaptation whereby cells are rendered resistant to injury by prior exposure to smaller doses of the same stress, which threatens to cause the injury in question. It is highly problematic to screen for potential therapeutics that cause the preconditioning response, since compounds that are identified as causing the preconditioning response generally also cause stress on the cell.
The accumulation of malfolded proteins in the endoplasmic reticulum leads to accumulation of reactive oxygen species. The protein kinase PERK has been shown to be activated by the stress of the accumulation of malfolded proteins in the endoplasmic reticulum (ER stress), and in turn phosphorylates the translation initiation factor eIF2α on its alpha subunit (Harding, H., Zhang, Y., and Ron, D. (1999). Translation and protein folding are coupled by an endoplasmic reticulum resident kinase. Nature 397, 271-274). A different eIF2α kinase, GCN2, has been also shown to phosphorylate eIF2α, however it acts in response to nutritional stress, not ER stress (Harding, H., Novoa, I., Zhang, Y., Zeng, H., Schapira, M., and Ron, D. (2000). Regulated translation initiation controls stress-induced gene expression in mammalian cells. Mol. Cell 6, 1099-1108). eIF2α phosphorylation leads to marked reduction in protein biosynthesis (Harding, H., Zhang, Y., Bertolotti, A., Zeng, H. and Ron, D. (2000). Perk is essential for translational regulation and cell survival during the unfolded protein response. Mol. Cell 5, 897-904) and to the expression of a transcription factor, ATF4, which then activates stress response genes in a signaling pathway termed the Integrated Stress Response (Harding, H., Novoa, I., Zhang, Y., Zeng, H., Schapira, M., and Ron, D. (2000). Regulated translation initiation controls stress-induced gene expression in mammalian cells. Mol. Cell 6, 1099-1108). This activation pathway is down-regulated by the activity of a phosphatase holoenzyme that dephosphorylates eIF2α on serine 51 (in yeast eIF2α, corresponding to residue 52 in rodents or humans). The phosphatase holoenzyme consists of the catalytic subunit of protein phosphatase 1 (PP1c) and GADD34, an eIF2α-specifc regulatory subunit of the phosphatase (Novoa, I.; Zeng, H., Harding, H., and Ron, D. (2001). Feedback inhibition of the unfolded protein response by GADD34-mediated dephosphorylation of eIR2α. J. Cell Biol., 153, 1011-1022).
This invention involves the discovery that the activation of stress response genes in the integrated stress response promotes resistance to both the stress of malfolded proteins in the endoplasmic reticulum and to the consequences of the accumulation of ROS. Therefore, the activation pathway of the integrated stress response pathway provides a desirable target for screening test substances capable of activating the pathway to promote preconditioning. Furthermore, screening test substances through the integrated stress response provides the advantage of identifying compounds, which activate the pathway, yet do not provide stress.